Project summary Golgi-derived microtubules (GDMTs) are a subset of microtubules (MTs) that form at the Golgi membrane. GDMTs are important for Golgi integrity and positioning as well as intracellular trafficking. A small number of centrosomal and MT-associated proteins have been implicated in GDMT nucleation, but how this machinery is regulated in physiological context is unknown. The MT network that serves for the intracellular transport and positioning of secretory vesicles is especially important in pancreatic ?-cells, which secrete insulin in response to glucose. Our lab has previously shown that the dense MT network in ?-cells acts as a negative regulator of insulin secretion, and is destabilized upon glucose stimulation to allow for efficient insulin release. However, glucose stimulation also increases new GDMT nucleation at the same time that preexisting MTs are lost to destabilization. It is not known how glucose signaling leads to the GDMT nucleation increase, or what role these newly formed MTs play in ?-cells. My preliminary data in cultured cells show that the GDMT increase involves cAMP signaling through its effector EPAC2. I hypothesize that EPAC2 can directly increase GDMT nucleation through its interaction with the MT stabilizer LC1. My data also show that the kinetics of the GDMT nucleation increase in high glucose mirrors the GSIS kinetics curve, and indicate a role for GDMTs in insulin granule biogenesis at the Golgi. Based on these data, I hypothesize that the GDMTs support timely replenishing of the insulin granule pool while it is depleted during secretion. Specifically, GDMTs could serve as tracks for the ER-to-Golgi transport of proinsulin and/or promote budding of the trans-Golgi to form insulin granules and their movement away from the Golgi area. It is also possible that disturbance of GDMT nucleation plays a role in type 2 diabetes formation and progression, causing the uncontrolled increase in MT density seen in this condition which prevents proper trafficking and processing of insulin. In Aim 1, I propose to determine whether EPAC2-dependent regulation of GDMTs is physiologically relevant, utilizing mouse and human islets. Next I will determine which domain of EPAC2 is involved in its regulation of GDMTs, and test whether this regulation is through its known interactor, LC1 or its Rap GEF activity. In Aim 2, I propose to determine GDMT functions in ?-cells, utilizing a number of approaches that have been found to decrease the GDMT population in other cell types. First, I will determine how GDMT depletion will influence MT network in ?-cells, their density, directionality, and stability. Next, I will determine whether GDMT defects affect GSIS and/or proinsulin trafficking and processing. Finally, I will utilize islets from diabetic mice and human donors to determine if GDMTs are changed in this condition.